Treatment for patients with metastatic melanoma is inadequate. A subset of metastatic melanoma patients can undergo meaningful tumor regression in response to agents that modify lymphocyte activation and/or expansion, for example, IL-2, anti-CTLA4, or IL-2 in combination with transfer of ex vivo expanded tumor-infiltrating lymphocytes (TIL). However, most patients receiving these therapies fail to respond or to achieve lasting benefit. Preclinical studies conducted in our laboratories and confirmed by other investigators provide strong evidence that inhibition of TGF-beta signaling can markedly enhance the anti-tumor activity of CD8+ cytotoxic T-lymphocytes (CTL) in animal models. We propose to extend these studies to determine optimal approaches for clinical development of agents that inhibit TGF-beta signaling in combination with IL-2, IL-2 + TIL, anti-CTLA4, or other related immunotherapeutic manipulations. The goal of the clinical trials proposed in this application is to improve the rate and quality of tumor responses in patients with metastatic melanoma and to reduce the morbidity and mortality from this disease. In Aim 1 we propose to confirm the improved anti-tumor effects of CD8+ TGFRII-DNR cells (CD8+ lymphocytes with the transgene for the dominant negative transforming growth factor beta receptor II) in mouse models and to determine if the anti-tumor activity of CD8+ TGFRII-DNR cells can be improved by addition of IL-2, addition of anti-CTLA4, and/or addition of anti-CD137. Furthermore, we propose to further characterize the mechanisms by which TGF-betaattenuates anti-tumor lymphocyte responses, in particular, to determine if the effects of TGF-beta are directly on CD8+ CTL or indirectly through induction of Treg (T regulatory cells which inhibit CD8+ and CD4+ anti-tumor lymphocyte responses), to determine the role of tumor driven TGF- beta in induction of Treg, and to determine whether and how Treg attenuate CD8+ CTL mediated tumor rejection. In Aim 2, we propose to create a retroviral vector carrying the TGFRII-DNR gene suitable for use in human clinical trials, to develop methods for transduction and expansion of human melanoma-derived TIL, and to characterize the cell product. We will also use mouse melanoma models to determine the optimal conditions necessary to maximize the anti-tumor effects of TGFRII-DNR CTL, and will use the results of the experiments to guide the design of a clinical trial. In Aim 3, we propose to evaluate non-genetic (pharmacologic) approaches to inhibit TGF-beta combined with an immunotherapy. Finally, in Aim 4, we propose to conduct proof of concept clinical trials in which TGF-beta inhibition is combined with an immune therapy. We propose that one of the clinical trials will involve adoptive transfer of melanoma TIL carrying the gene for TGFRII-DNR.